The Genotype®MTBDRplus molecular test is a method that allows identifcation of the most frequent mutations associated with resistance to major frst-line antituberculosis drugs, Isoniazid (INH) and Rifampicin (RfP). The aim of this study was to evaluate the performance of the molecular test with culture and smear- positive sputum samples. We evaluated 95 cultures and 100 sputum samples with resistance profles previously determined by the reference method "Agar Plate Proportions" (APP). The molecular test from cultures showed a sensitivity of 100%, 97,5% and 96,97% for RIf, INH and MDR respectively while from sputums the sensitivity was 95,65%, 96,77% and 95,24% for RIf, INH and MDR respectively. We conclude that the molecular test Genotype®MTBDRplus is a very useful tool to detect resistance to isoniazid and rifampicin simultaneously (MDR-TB) in up to 72 hours from sputum samples or cultures.